Non-Native Marine Macroalgae of the Azores: An Updated Inventory

: Non-native species (NNS) represent a threat to biodiversity, and their occurrence and distribution should be periodically updated and made easily available to researchers and policymakers. An updated inventory of macroalgal NNS currently present in the Azores was produced based on published reports. Data concerning the ﬁrst report and the distribution in the archipelago are provided for each species, as well as their respective native ranges and possible vectors of introduction. The resulting list comprises 42 taxa, i


Introduction
The presence of Non-Native Species (NNS), i.e., taxa that are introduced or facilitated by human activities to a given area, is considered one of the major threats to global biodiversity, the economy, and public health [1,2].Since algae have an important role in the marine ecosystem, an algal invasion changes key characteristics such as community abundance, resource allocation, nutrient acquisition rates, and competition mechanisms [2].Research and policy activities related to NNS require that current and historical data, including species status, introduction pathway, and degree of establishment, are accessible and useful [3].
The Azores is a remote archipelago in the North Atlantic, comprising nine main volcanic islands and several islets, separated into three groups (Figure 1).These islands have a relatively recent origin and are spread over 500 km from east to west [4].The warm Gulf Stream, acting as a thermal buffer, contributes to the moderate sea water temperatures in the Azores, which in turn influences its climate [5].Despite its high latitude, the archipelago's climate is subtropical, with moderate temperatures, low insolation rates, regular and abundant rainfall, and vigorous winds [6].The marine flora of the Azores presents connections with subtropical and tropical Atlantic America [7] but mainly with the Eastern Atlantic, the Mediterranean, and other Macaronesian islands [8].
connections with subtropical and tropical Atlantic America [7] but mainly with the Eastern Atlantic, the Mediterranean, and other Macaronesian islands [8].In 2006, Cardigos et al. [9] reported 33 introduced species (12 macroalgae and 21 invertebrates) in the first inventory of non-native species in the Azores.Later, updated inventories were published (e.g., [10,11]), as well as other isolated reports (e.g., [12][13][14]).Castro et al. [11] registered 66 NNS in the Azores, 22 of which are macroalgal species, the most representative group of the archipelago.In fact, the Azores were reported to have double the global ratio of non-native macroalgal species [10].Hence, this archipelago poses as a crossroad for native species distribution [15] but also for non-native species introductions [11].
The current study aims to produce an updated inventory of the non-native macroalgal species reported for the Azores and their respective introduction histories based on data from the literature.The updated list increases the number of macroalgal NNS reported for the archipelago, corrects taxonomic changes or conflicts, and amends previously reported distributions, representing a support tool for research and policymaking.

Materials and Methods
The study area comprises the nine Azorean islands and Formigas islets (Figure 1), here included due to the relevance of the marine flora found in their shallow banks [16].The present inventory includes alien, non-indigenous, introduced, and non-native species, i.e., species currently present but with native distribution ranges other than the Azores, reported in published scientific articles, monitoring programs, technical reports, and Ph.D. theses.Cryptogenic species, i.e., those whose statues that are not clearly native or non-native [9,10], and those classified with uncertain status [16][17][18][19][20][21][22][23][24] in the archipelago, were not included.The criteria used by the mentioned authors to classify the species as non-native were not evaluated; therefore, their statuses are based on the reported data.Nevertheless, a few decisions were made regarding conflicting reports: 1-Asparagopsis taxiformis is kept as an NNS despite the suggestion of Neto et al. (2021) to change its status to native, and this study agrees with other publications based on distributional and molecular data (e.g., [10,11,25]); 2-Antithamnion nipponicum and Corynomorpha prismatica were reincluded in the NNS list since Castro et al. [11] based their removal only on their sporadicity; 3-records of Lophocladia trichocladus were removed from Faial, Codium fragile In 2006, Cardigos et al. [9] reported 33 introduced species (12 macroalgae and 21 invertebrates) in the first inventory of non-native species in the Azores.Later, updated inventories were published (e.g., [10,11]), as well as other isolated reports (e.g., [12][13][14]).Castro et al. [11] registered 66 NNS in the Azores, 22 of which are macroalgal species, the most representative group of the archipelago.In fact, the Azores were reported to have double the global ratio of non-native macroalgal species [10].Hence, this archipelago poses as a crossroad for native species distribution [15] but also for non-native species introductions [11].
The current study aims to produce an updated inventory of the non-native macroalgal species reported for the Azores and their respective introduction histories based on data from the literature.The updated list increases the number of macroalgal NNS reported for the archipelago, corrects taxonomic changes or conflicts, and amends previously reported distributions, representing a support tool for research and policymaking.

Materials and Methods
The study area comprises the nine Azorean islands and Formigas islets (Figure 1), here included due to the relevance of the marine flora found in their shallow banks [16].The present inventory includes alien, non-indigenous, introduced, and non-native species, i.e., species currently present but with native distribution ranges other than the Azores, reported in published scientific articles, monitoring programs, technical reports, and Ph.D. theses.Cryptogenic species, i.e., those whose statues that are not clearly native or nonnative [9,10], and those classified with uncertain status [16][17][18][19][20][21][22][23][24] in the archipelago, were not included.The criteria used by the mentioned authors to classify the species as non-native were not evaluated; therefore, their statuses are based on the reported data.Nevertheless, a few decisions were made regarding conflicting reports: 1-Asparagopsis taxiformis is kept as an NNS despite the suggestion of Neto et al. (2021) to change its status to native, and this study agrees with other publications based on distributional and molecular data (e.g., [10,11,25]); 2-Antithamnion nipponicum and Corynomorpha prismatica were reincluded in the NNS list since Castro et al. [11] based their removal only on their sporadicity; 3-records of Lophocladia trichocladus were removed from Faial, Codium fragile from Graciosa, Asparagopsis taxiformis from São Jorge and Corvo, and Grateloupia turuturu (reported as G. filicina) from Santa Maria, Graciosa, Terceira, São Jorge, and Flores, after verification of the references given by Castro et al. [11].The conservative aspect of such decisions is based on the confirmation of species identifications during the exhaustive works of Neto et al. [16][17][18][19][20][21][22][23][24].Scytosiphon dotyi, for example, was collected twice in 30 years, with a 15-year gap [26]; Antithamnionella ternifolia remained a single record for 17 years [10], with multiple specimens found afterwards [24,27].Native distribution of macroalgal NNS and possible vectors are given as reported by each respective author.
The framework, taxonomy, nomenclature, and authorities in this checklist are built upon the current synthesis of taxa in AlgaeBase [28].It is beyond the scope of this paper to provide a rigorous assessment of the misapplication of species and genus names reported for the Azores without a critical examination of the macroalgal vouchers from the region.
For statistical purposes, native distribution was grouped as Pacific Ocean, Western Pacific, Northwestern Pacific, Indo-Pacific, Indian Ocean, Northeastern Atlantic, and Western Atlantic.Hull fouling and ballast water were grouped as 'shipping', while aquaculture/aquarium were grouped as 'escape from confinement'.Natural dispersal mechanisms such as rafting were not considered when analyzing the introduction vectors.When the first report of a species is given as a period of years, only the first year is considered.

Species Invetory
The resulting list comprises a total of 42 non-native macroalgal species (8.05% of the Azorean marine flora of 522 taxa), with the correction of previously reported distributional ranges based on taxonomy and the literature review (Table 1, Supplementary Material File S1).
Table 1.Inventory of the non-native macroalgal species currently reported for the Azores, including the respective native distribution, possible introduction vector to the Azores, distribution per island, and year of first report in the archipelago.Taxa indicated with a "+" denotes additions to the previous inventory listed in Castro et al. [11].Taxa with distribution restricted to a single island are indicated with "*".Taxa based on a single record are indicated with " • ".São Miguel [14]; Faial [14] Ballast waters, Hull fouling [14] 2019 [14] Order Ectocarpales Family Chordariaceae

Taxon Native Range Distribution in the Azores Possible Vector First Record for the Azores
Family Codiaceae

Codium fragile
Northwestern Pacific [10] São Miguel [7,10,11,24]; Santa Maria [11,22]; Graciosa [21]; Pico [20]; Flores [23]; Corvo [7,10,11,23] Ballast water, Hull fouling [10] 1993 [7] Family Halimedaceae Halimeda incrassata * Western Atlantic and Indo-Pacific [55] Santa Maria [11,48] Hull Fouling [48] 2016 [48] 1 The species is established in the Mediterranean where it was introduced from shellfish aquaculture [45]. 2 Castro and Viegas [46] reported the species Antithamnion sarniensis, with Neto [42] further amending this record as the heterotypic synonym Antithamnionella spirographidis.Since Antithamnion sarniensis is currently regarded as a homotypic synonym of Antithamnionella ternifolia [28], the record is only considered for the latter species. 3The species is established in the Atlantic coast of France where it was introduced from shellfish aquaculture [44,56]. 4Molecular-based identification is needed to confirm this record since the true Lophocladia trichoclados is apparently an Atlantic species [47], therefore not an NNS. 5 Xiphosiphonia pennata is a complex species reported in the Atlantic and the Indo-Pacific.Molecular-based identification is needed to confirm this record, since the true X. pennata is apparently restricted to European shores [51], therefore probably not an NNS. 6Xiphosiphonia pennata is a complex species (including X. pinnulata) reported in the Atlantic and the Indo-Pacific.Molecular-based identification is needed to confirm this record, since the true X. pinnulata is apparently restricted to the Atlantic [51], therefore probably not an NNS. 7According to Gavio and Fredericq [57], the Atlantic non-native Grateloupia doryphora should be referred to as G. turuturu; therefore, the distribution in the Azores is based only on reports of the first species.All reports based on G. filicina (since [58]) are removed from the previous inventory [11].Nevertheless, vouchers of this species reported to the Azores should be re-examined, since reports of G. filicina var.luxurians, currently referred as G. subpectinata, are thought to be introductions in Ireland and Britain [59].

Species Composition
The updated list is composed of (Figure 2a): • Five brown algae (5% of the Ochrophyta reported in the Azores); • Thirty-three red algae (9% of the Rhodophyta reported in the Azores); • Four green algae (5% of the Chlorophyta reported in the Azores); • Therefore, 12% of the non-native macroalgal species belong to the Ochrophyta, 79% to the Rhodophyta, and 9% to the Chlorophyta.
Diversity 2023, 15, x FOR PEER REVIEW 7 of 14 • Therefore, 12% of the non-native macroalgal species belong to the Ochrophyta, 79% to the Rhodophyta, and 9% to the Chlorophyta.
Within the three macroalgal Phylla, the most represented orders are (Table 1): • Four of the five (80%) Ochrophyta species belong to the Ectocarpales, characterized by isomorphic gametophyte and sporophyte composed of uniseriate, branched, or unbranched filaments [28]; • 25 of the 33 (76%) Rhodophyta species belong to the Ceramiales, an order characterized by isomorphic gametophyte and sporophyte of widely varying morphology of uniaxial structure [28]; • All four (100%) Chlorophyta species belong to the Bryopsidales, an order characterized by simple to complex siphonous thalli [28].

General Distribution
All islands host non-native macroalgal species, with the following percentage in relation to the reported marine flora (Figure 2b):

•
The most isolated islands in the Western Group (Flores and Corvo) present lower percentages of NNS (6.25% and 4.25%, respectively); The islands with the most used marina for transatlantic recreational sailing (São Jorge Within the three macroalgal Phylla, the most represented orders are (Table 1): • Four of the five (80%) Ochrophyta species belong to the Ectocarpales, characterized by isomorphic gametophyte and sporophyte composed of uniseriate, branched, or unbranched filaments [28]; • 25 of the 33 (76%) Rhodophyta species belong to the Ceramiales, an order characterized by isomorphic gametophyte and sporophyte of widely varying morphology of uniaxial structure [28]; All four (100%) Chlorophyta species belong to the Bryopsidales, an order characterized by simple to complex siphonous thalli [28].

General Distribution
All islands host non-native macroalgal species, with the following percentage in relation to the reported marine flora (Figure 2b):

•
The most isolated islands in the Western Group (Flores and Corvo) present lower percentages of NNS (6.25% and 4.25%, respectively);

•
The islands with the most used marina for transatlantic recreational sailing (São Jorge and Faial) present higher percentages of NNS (12.90% and 16.87%, respectively).

Specific Distribution (Maps in Supplementary Material File S2)
The presence of 13 species is restricted to single islands, namely: • Antithamnionella elegans, Antithamnionella ternifolia, Corynomorpha prismatica, Grallatoria reptans, and Gymnophycus hapsiphorus in São Miguel; The distribution of six species is restricted to the different Groups of islands, namely: • Antithamnion hubbsii, Lophocladia trichoclados, and Scytosiphon dotyi in the Eastern Group; • Aglaothamnion cordatum, Laurencia chondrioides, and Laurencia minuta in the Central Group; No species is exclusively found in the Western Group.
Asparagopsis armata is the only species reported from all islands of the archipelago.

Native Range
The original distributional ranges of the non-native macroalgal species of the Azores are (Figure 3a):

•
7% from the Pacific Ocean, 7% from the Western Pacific, and 17% from the Northwestern Pacific; • 31% from the Indo-Pacific; • 17% from the Indian Ocean; • 2% from the Northeastern Atlantic, and 19% from the Western Atlantic.

Introduction Vectors
The possible vectors of introduction (based on published reports) for the non-native macroalgal species in the Azores are (Figure 3b):

History of First Reports
The cumulative number of non-native macroalgal species shows the following trend (Figure 4):

Introduction Vectors
The possible vectors of introduction (based on published reports) for the non-native macroalgal species in the Azores are (Figure 3b):

History of First Reports
The cumulative number of non-native macroalgal species shows the following trend (Figure 4):

Discussion
The list of non-native macroalgal species in the Azores is here increased to 42 species 20 more species than in the last inventory by Castro et al. [11].This expansion includes th reintroduction of species previously recorded by Micael et al. [10] as well as additiona

Discussion
The list of non-native macroalgal species in the Azores is here increased to 42 species, 20 more species than in the last inventory by Castro et al. [11].This expansion includes the reintroduction of species previously recorded by Micael et al. [10] as well as additional species reported as non-native by other authors in the last five years (e.g., [12][13][14]21,24]).Since the extensive study by Micael et al. [10], the known marine flora from the Azores encompasses 83 more species (439 to 522, [26]), including 16 more NNS.Nevertheless, the newly reported macroalgal NNS increases the ratio that Micael et al. [10] reported from 6 to 8% of the Azorean marine flora, rising from double the global percentage (3%) to almost triple.
The ratio of non-native species belonging to Rhodophyta has also increased from that reported by Micael et al. [10], namely from 65 to 79%, and is now higher than the global pattern [60].As discussed by Micael et al. [61], red algae appear to be particularly efficient in introduction and invasive events.Their success can be attributed to the combination of various factors, including: 1-the ability to reproduce by fragmentation, increasing the potential pool of propagules [62]; 2-the ability to use the entire light spectrum, enabling them to establish in shadow environments [63]; and 3-their high bromophenol content which might help to deter predators [64].
Despite the low representation of the Chlorophyta in the present inventory, all the green algae reported as NNS in the Azores belong to the Bryopsidales.Members of this order present rapid growth from fragmented thalli (e.g., [65]), and many taxa became invasive out of their native range (e.g., [66]).In fact, Caulerpa webbiana was classified as one of the species to be prioritized for monitoring and eradication measures in the Azores [67].
Likewise, although only five species of brown algae are reported in the updated inventory, it includes Rugulopteryx okamurae, an extremely invasive species with high propagation capacity [68] This species has caused major impacts in communities where it has invaded (e.g., [69]), and has quickly made its way to the European list of invasive non-native species [70].R. okamurae is also the only macroalga with specific legislation for its monitorization and control in the Azores [71].
The ratio of non-native macroalgal species with native distribution in the Pacific Ocean (31%) and the Indo-Pacific (31%) remains equal to those reported by Micael et al. [10], even with the now increased number of NNS (from 26 to 42 species).Instead, the ratio of species from the Indian Ocean decreased (23% to 17%), and the ratio from the Atlantic Ocean increased (15% to 21%).The main difference is the increase in species from the Western Atlantic (11% to 19%), a trend also observed in other Macaronesian archipelagos, namely Madeira (e.g., [72]) and the Canary Islands (e.g., [73]).NNS originally distributed in the Atlantic Ocean might include species that reached the archipelago by rafting in the dominant Atlantic currents, with their subsequent establishment facilitated by the increase in seawater temperatures [13,72].Although not directly introduced by human activities, the growing accumulation of marine litter facilitates new introductions by offering new habitats for drifting macroalgae [74].
The updated distribution of non-native macroalgal species in the Azores shows that previously reported species [9,10] have spread to other islands.This rise is probably due to further transport between harbors and marinas but may also result from additional efforts to identify non-native species [75].
No experimental or empirical evidence of introduction vectors for the non-native macroalgal species currently found in the Azores has been found.Nevertheless, different authors reporting on those species suggested possible vectors for their introductions (see detailed reports in Section 3.1).Transport via shipping is the most representative possible vector, which probably contributed to 68% of the non-native macroalgal species reported.This ratio is similar to that reported by Micael et al. [10], even though those authors studied fewer species than the present work.This possible ratio is higher than that reported for marine species in other European waters [76], mostly due to the low representation of other vectors (such as artificial channels and aquaculture).The introduction vector of 27% of the NNS currently present in the Azores is still unknown, and further studies are necessary to identify other possible existing vectors.
Although studies of the marine flora of the Azores started in 1844 [77], the first record of a non-native macroalgal species dates from 1928 [39].This date coincides with the second wave of marine species introductions that happened at the beginning of the 20th century, associated with the increase in maritime traffic [78].Two surges in first reports of NNS were observed in this study and coincided with increased sampling efforts in the Azores.In the late 1980s, the Biology Department of the University of the Azores organized various scientific expeditions (Graciosa/88, Faial e Pico/89, Flores e Corvo/89, Santa Maria e Formiga/90), which resulted in the report of nine new NNS.[79], which included the level of NNS introduced by human activities as a qualitative descriptor, new records of non-native macroalgal species have been steadily reported.
Further taxonomic work is necessary to correct possible misidentifications in previous reports (e.g., [16][17][18][19][20][21][22][23][24]).Consequently, many species names may not be accurate because the taxonomic concepts of previous reports were based mostly on morphological similarities without comparative molecular systematics (with a few exceptions such as [13,14]).Species distribution in the Azores might also be underrepresented, especially in islands where sampling is sporadic [22] or in habitats not specifically investigated [26].Therefore, further investigation is still needed.Additionally, the invasiveness risk of NNS should be assessed to support policymakers [80,81] in preventing, minimizing, or mitigating their impact in a cost-efficient manner [76].
Nevertheless, this list contributes to estimating the presence of non-native species throughout the Azores, providing updated and accessible data for invasiveness risk assessments and subsequent policymaking [3].

Figure 1 .
Figure 1.The archipelago of the Azores and its location in the North Atlantic (box).

Figure 1 .
Figure 1.The archipelago of the Azores and its location in the North Atlantic (box).

Figure 2 .
Figure 2. Non-native macroalgal species in the Azores: (a) Composition of the Azorean marine flora in relation to the species' native status and composition of the non-native species (NNS) in relation to the main groups of algae; (b) Number of macroalgal non-native species (NNS) and its percentage in relation to the reported marine flora, for each island of the Azores.

Figure 2 .
Figure 2. Non-native macroalgal species in the Azores: (a) Composition of the Azorean marine flora in relation to the species' native status and composition of the non-native species (NNS) in relation to the main groups of algae; (b) Number of macroalgal non-native species (NNS) and its percentage in relation to the reported marine flora, for each island of the Azores.

•Figure 3 .
Figure 3. Introduction of the non-native macroalgal species in the Azores: (a) Native range of the reported species; (b) Possible vectors of introduction.

•
First NNS reported in 1928; • First surge of NNS reports in the late 1980s; • Second surge of NNS reports in the mid-2000s; • Steady increase in the number of NNS reports in the last 15 years.

Figure 3 .
Figure 3. Introduction of the non-native macroalgal species in the Azores: (a) Native range of the reported species; (b) Possible vectors of introduction.

• 1 Figure 4 .
Figure 4.The cumulative number of non-native macroalgal species, according to their year of record in the Azores.

Figure 4 .
Figure 4.The cumulative number of non-native macroalgal species, according to their year of record in the Azores.